1. Field of the Invention
The invention is directed to a sample holder for a microscope. A sample holder of this kind comprises a sample chamber which is filled with an immersion liquid and in which a sample is located. The sample chamber has an upper opening. Further, the sample holder comprises means for translating the sample relative to a detection objective of the microscope and means for rotating the sample around an axis of rotation extending in a substantially horizontal plane which encloses an angle other than zero degrees with the optical axis of the detection objective.
The sample holder according to the invention can be applied particularly in connection with single plane illumination microscopy (SPIM) and selective plane illumination microscopy. Whereas in confocal laser scanning microscopy the sample is scanned point by point in a plurality of planes at different depths and three-dimensional image information about the sample is obtained from this, the SPIM technique is based on widefield microscopy and makes it possible to generate three-dimensional images of samples based on optical sections through different planes of the sample.
The advantages of SPIM include faster acquisition of images, reduced bleaching out of biological samples, and an expanded depth of penetration of the focus in the sample.
2. Description of Related Art
Basically, in the SPIM technique fluorophores which are contained in the sample or introduced into the sample are excited by laser light which is shaped as a light sheet or which is guided over the sample in such a way that the shape of a light sheet results in effect (i.e., over the period of observation). Each light sheet illuminates a plane in the depth of the sample, and an image of the sample in this plane is obtained by means of this illumination. It is important that elements in the light sheet plane are projected on the detector plane or that the light sheet plane and detector plane are conjugate with respect to one another. In conventional microscope constructions in which the detector plane extends perpendicular to the optical axis of the detection beam path, the direction in which the light is detected is perpendicular or at least virtually perpendicular to the plane of illumination.
SPIM technology is described, for example, in Stelzer et al., Optics Letter 31, 1477 (2006), Stelzer et al., Science 305, 1007 (2004), DE 102 57 423 A1, and WO 2004/0530558 A1.
The disclosure of these publications includes a sample holder which makes it possible to orient the sample in an optimal manner with a view to obtaining three-dimensional image data from different viewing directions. For this purpose, the sample is embedded in a gel which has been shaped to form a circular cylinder, and this gel cylinder is introduced into a sample chamber filled with an immersion medium, for example, water. The refractive index of the gel must not differ substantially from the refractive index of the surrounding immersion medium.
The gel cylinder enclosing the sample is positioned in the sample chamber in such a way that its axis of rotation extends in the direction of gravitational force, which has advantages for the positioning of the sample in view of the deformability of the gel. It is supported in such a way that it can be displaced in translation and optionally also rotated around its axis of rotation for image recording inside the sample chamber.
The optical axis of the detection objective which collects the detection light coming from the sample is oriented approximately perpendicular to the axis of rotation of the gel cylinder and accordingly does not extend vertically as in the typical microscope construction, but rather horizontally (i.e., perpendicular to the direction of gravitational force).
For image recordings which must be obtained with a large imaging scale and high numerical aperture, detection objectives constructed as immersion objectives are generally used. The immersion objectives project through a wall of the sample chamber into the sample chamber and are sealed at their outer circumference relative to the wall of the sample chamber in order to prevent the immersion medium from running out at the location where the objective penetrates.
A considerable disadvantage of this construction consists in that a special construction of the microscope is required for horizontally aligned detection beam paths because standard microscopes operate with a vertical detection beam path. If other contrast methods besides SPIM are to be used, the special microscope construction must be outfitted correspondingly at increased cost. Further, when immersion objectives are used, it is extremely difficult to change the detection objective because of sealing. A similar construction is described in Voie et al., Hearing Research 171, 119 (2002) and in Voie et al., J. of Microscopy 170, 229 (1993). Both horizontal and vertical detection beam paths are described. The axis of rotation around which the sample is rotated is aligned horizontally. The rotary drive axle projects laterally into a sample chamber fashioned from polyacetal which also contains the sample and a suitable ambient liquid. The sample is held in the chamber at this axle and is surrounded on all sides by the ambient liquid with the exception of the connection to the axle. The sample chamber is sealed on all sides but can also be open at the top in case of a vertical orientation of the detection beam path. However, the lens of the detection objective does not contact the immersion liquid in this case. A translation of the sample is achieved through movement of the entire chamber. No further details are given with respect to holding the sample with a horizontal axis of rotation.
A sheet-shaped illumination of the sample is also described in U.S. Pat. No. 3,398,634, wherein a conventional microscope stand is used. Rotation of the sample is not described.
U.S. Pat. No. 5,680,484 describes a sample holder in the form of a transparent cylinder which is filled with a liquid and which is held at two locations along its circumference and is set in rotation by a belt drive. However, it is relatively laborious to clamp a glass cylinder of this type in the device and to change the sample in such a glass cylinder. Means for translation of the sample are not described.